Systems for tightly regulated gene expression

a gene expression and tightly controlled technology, applied in the field of tightly controlled bacterial expression vectors, can solve the problems of leakage of expression systems, compromising the regulation of cloned inserts, and unwanted background expression of genes

Inactive Publication Date: 2005-08-18
CONJUGON
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0016] The present invention further provides a method, comprising providing a gene of interest inserted into a vector comprising transcription terminators and a low copy number origin of replication; and expressing the gene of interest in a bacterial host. In some embodiments, the gene of interest encodes a toxic protein or RNA. In other embodiments, the gene of interest encodes a protein with a toxic metabolite. In preferred embodiments, the gene of interest is maintained in the vector under growth conditions and the protein (e.g., a toxic protein) accumulates in the bacterial host.
[0017] The present invention is not limited to particular transcription terminators. In some preferred embodiment, the transcription terminators comprise rrnB r

Problems solved by technology

While these systems are commonly used and contain many desirable features, these expression systems are subject to leaky expression from the promoters, which can prohibit cloning of extremely toxic proteins, RNA, or enzymes producing toxic metabolites.
While these bacterial and phage systems offer the ability to express a gene at high levels of expression, they often suffer from unwanted background expression of the gene.
Second, the majority o

Method used

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  • Systems for tightly regulated gene expression
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Examples

Experimental program
Comparison scheme
Effect test

example 1

Plasmid Construction

[0098] This Example describes the construction of exemplary plasmids of the present invention. Table 1 shows the names and corresponding Figure and SEQ ID NO designations for the plasmids described below. Sequences of plasmids and selected vector elements are shown in FIG. 11.

TABLE 1PlasmidsNameFigure (depicting map)SEQ ID NOpCON3-86B21pCON7-7432pCON7-7143pCON5-2554pCON7-7765pCON7-5876pCON4-4287pCON7-1198

A. Materials and Methods

Bacterial Strains and Media

[0099] The Escherichia coli strain utilized was NovaBlue {endA1 hsdR17(rK12-mK12+) supE44 thi-1 recA1 gyrA96 relA1 Δlac F′(proA+B+lacIqZAM15::Tn10 (TcR))} from Novagen (Madison, Wis.). All cloning was performed using standard methods known in the art, and using Luria Bertani growth media supplemented with 50 μg / ml kanamycin to permit selection for plasmids. For cloning of toxic gene products such as the colicins, the growth media was supplemented with 0.8% glucose to further repress the lactose promoter.

...

example 2

Gene Expression

[0108] This example describes the measurement of levels of expression from the vectors described in Example 1.

[0109] Using the standard assay for β-galactosidase activity, the promoter activity for vectors pCON3-86B, pCON5-25, pCON7-74, and pCON7-77 were obtained in repression conditions (Luria-Bertani broth supplemented with 0.8% glucose and 50 μg / ml kanamycin) and expression conditions (Luria-Bertani broth supplemented with 1 mM IPTG and 50 μg / ml kanamycin). Cultures were assayed in duplicate at an OD600 nm of 0.3-0.5 and expressed as Miller Units. The results are shown in FIG. 10.

[0110] As observed in FIG. 10, the promoter activities of pCON5-25 and pCON7-77 in repression medium are not significantly different from vectors pCON3-86B and pCON7-74, which do not contain the gene for α-galactosidase. However, upon de-repression with 1 mM IPTG, the promoter activity of pCON5-25 (with modified-pSC101 origin) is increased approximately 50-fold and the activity of pCON7...

example 3

Expression of Toxic Proteins

[0111] The vectors of the present invention were used to clone and stably maintain the genes encoding colicins D (pCON7-58), E3 (pCON4-42), E7 (pCON7-11), E3 (pCON12-82) in the absence of the cognate immunity proteins, with the ability to achieve high levels of protein / RNA expression upon de-repression of the promoter.

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Abstract

The present invention relates to bacterial expression vectors. In particular, the present invention provides tightly-regulated bacterial expression vectors designed for the cloning and expression of toxic proteins, RNA, and metabolites in vivo. The present invention thus provides methods of expressing protein and RNAs that were previously not able to be expressed.

Description

[0001] This application claims priority to Provisional Patent Application Ser. No. 60 / 529,255, filed Dec. 12, 2003, which is incorporated herein by reference in its entirety.FIELD OF THE INVENTION [0002] The present invention relates to bacterial expression vectors. In particular, the present invention provides tightly-regulated bacterial expression vectors designed for the cloning and expression of toxic proteins, RNA, and metabolites in vivo. BACKGROUND OF THE INVENTION [0003] Although many prokaryotic expression systems have been developed for expression of recombinant proteins, most gene expression systems in gram-negative bacteria such as Escherichia coli have relied exclusively on a limited set of bacterial promoters. The most widely used bacterial promoters have included the lactose (lac) (Yanisch-Perron et al. Gene 33: 103-109 {1985}), tryptophan (trp) (Tacon et al. Mol. Gen. Genet. 177:427-38 {1980}), and hybrid derivatives such as the tac (deBoer et al. Proc. Natl. Acad. S...

Claims

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Application Information

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IPC IPC(8): C07H21/04C12N1/21C12N15/11C12N15/63C12N15/70C12N15/74C12P21/02C12P21/06C12Q1/68
CPCC12N15/70
Inventor ANTHONY, LARRYFILUTOWICZ, MARCINSUZUKI, HIDEKI
Owner CONJUGON
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